This proposal opens new territory in the study of cholinergic synapses. Acetylcholine (ACh) is broken down in the synaptic cleft, and a requirement for its re-synthesis is choline uptake via the choline transporter (CHT). We will study the human choline transporter (hCHT) in a heterologous expression system with a view to understanding not only its role in the plasma membrane, but also in the ACh-containing synaptic vesicles (SVs) that traffic CHT to the plasma membrane. ACh is a major neurotransmitter in the human nervous systems that modulates cognitive processes such as attention, reward, arousal, sleep, learning, and memory. ACh is also essential for muscular control not only in skeletal muscle, but also in smooth muscle and cardiac muscle. Deficits in cholinergic neurotransmission contribute to Alzheimer's disease and schizophrenia, and diminished cholinergic signaling correlates with neuromuscular diseases, such as myasthenia and tardive dyskinetics. Thus, understanding a key player in cholinergic transmission, hCHT, is an important problem with broad implications. Interestingly, whereas presynaptic choline uptake is rate limiting for sustained ACh synthesis, 90% of CHTs are on ACh-containing SVs, which deliver CHTs to the plasma membrane upon stimulation. We propose a biophysical analysis of CHT relevant to its function on the plasma membrane and in SVs. Because CHTs can transport ACh, an open question is CHT activity in ACh SVs. Our methods include hCHT expression in Xenopus oocytes to measure choline uptake and choline-induced current using the two-electrode voltage-clamp (TEVC). Our data so far show that choline uptake and induced current are pH sensitive (pKa 7.4) and, at vesicular pH (5.5), choline uptake and current are abolished. Furthermore, CHT-specific hemicholinium-3 binding suggests H+ titration of a specific residue near the choline-binding site. These observations led us to the hypothesis that protons inactivate CHTs on ACh-SVs, which allows them to be there (for delivery) while at the same time preventing ACh and H+ leakage from the SV. We will test this hypothesis by investigating ACh transport through CHTs at various pH. We will also look for pH-sensitive residue(s) in hCHT, and use TEVC to study the pH-regulation mechanism of uptake and current. Finally, we will characterize the polymorphic variant in hCHT, I89V, which correlates with increased anxiety and reduced libido in humans. Acetylcholine is a neurotransmitter that carries messages for cognitive processes such as attention, reward, arousal, sleep, learning, and memory in our brain, and it is also is essential for neuromuscular control, including our arm/leg movements and heartbeat. After acetylcholine is released, for normal signaling to reoccur, choline must be taken up back into the nerve. This is accomplished by the choline transporter, whose unusual regulation or genetic mutations are related to various diseases including Alzheimer's disease, schizophrenia, choreics, and tardive dyskinetic. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page [unreadable] [unreadable]